Date Published: June 13, 2013
Publisher: Public Library of Science
Author(s): Nicolle Marshall, Lalita Priyamvada, Zachary Ende, John Steel, Anice C. Lowen, Christopher F. Basler.
Reassortment is fundamental to the evolution of influenza viruses and plays a key role in the generation of epidemiologically significant strains. Previous studies indicate that reassortment is restricted by segment mismatch, arising from functional incompatibilities among components of two viruses. Additional factors that dictate the efficiency of reassortment remain poorly characterized. Thus, it is unclear what conditions are favorable for reassortment and therefore under what circumstances novel influenza A viruses might arise in nature. Herein, we describe a system for studying reassortment in the absence of segment mismatch and exploit this system to determine the baseline efficiency of reassortment and the effects of infection dose and timing. Silent mutations were introduced into A/Panama/2007/99 virus such that high-resolution melt analysis could be used to differentiate all eight segments of the wild-type and the silently mutated variant virus. The use of phenotypically identical parent viruses ensured that all progeny were equally fit, allowing reassortment to be measured without selection bias. Using this system, we found that reassortment occurred efficiently (88.4%) following high multiplicity infection, suggesting the process is not appreciably limited by intracellular compartmentalization. That co-infection is the major determinant of reassortment efficiency in the absence of segment mismatch was confirmed with the observation that the proportion of viruses with reassortant genotypes increased exponentially with the proportion of cells co-infected. The number of reassortants shed from co-infected guinea pigs was likewise dependent on dose. With 106 PFU inocula, 46%–86% of viruses isolated from guinea pigs were reassortants. The introduction of a delay between infections also had a strong impact on reassortment and allowed definition of time windows during which super-infection led to reassortment in culture and in vivo. Overall, our results indicate that reassortment between two like influenza viruses is efficient but also strongly dependent on dose and timing of the infections.
Reassortment is the process by which viruses carrying segmented genomes exchange gene segments. The reshuffling of genetic material achieved through reassortment supports rapid production of variant viruses that can be markedly different, genotypically and phenotypically, from the parental strains. The more gradual process of genetic drift, resulting from errors in genome replication, and the process of reassortment come together to generate vast genomic diversity among influenza A viruses. It is this diversity that, in turn, permits the rapid evolution of influenza viruses and the generation of novel pandemic and epidemic strains.
We have evaluated, to our knowledge for the first time, the efficiency with which influenza A viruses undergo reassortment in the absence of fitness differences among parental and progeny genotypes. Our results show that reassortment is very efficient: where high rates of co-infection are achieved, high frequencies of reassortant genotypes are seen. These findings demonstrate that compartmentalization within the cell does not prevent extensive mixing of gene segments from two co-infecting viruses. It remains unclear whether this mixing occurs in the cytoplasm upon virus uncoating, in the nucleus during replication, during nuclear export and trafficking to the cell membrane, during the assembly process or throughout the virus life cycle. It is clear, however, that at least one stage of the life cycle allows for unrestricted exchange of gene segments.